Lenvatinib e7080

Human HCC cell lines HepG2, Hep3B and Huh-7 were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM-high glucose (Sigma-Aldrich, St Louis, MO, USA) supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/mL, Gibco^™, Gaithersburg, MD, USA) at 37 °C under a humidified 5% CO₂ atmosphere during passages no longer than 25 passage. For cell treatments, lenvatinib (E7080, Selleckchem, Houston, TX, USA), first-line drug approved against advanced HCC [2 (link)], was employed at different concentrations from 0.5 to 30 µM. EG00229 trifluoroacetate was used as an antagonist of NRP1 activity, 300 µM cycloheximide (CHX) as protein synthesis inhibitor, 30 µM MG132 as proteasome inhibitor and 100 nM bafilomycin A1 as specific inhibitor of autophagy flux. All these reagents were acquired from Tocris Bioscience (Bristol, UK). Finally, CoCl₂ (Panreac AppliChem, Barcelona, Spain) was used as hypoximimetic at 100 µM to induce an in vitro hypoxic microenvironment.

FITC-conjugated mouse anti-human CD34 and mouse IgG1 K isotype control-FITC were from eBioscience (CA, USA). APC-conjugated mouse anti-human VEGFR-3 and mouse IgG1 isotype control-APC were from R&D systems (USA). Anti-LYVE-1 antibody was from Abcam. FITC-conjugated secondary antibodies were from KeyGEN Biotechnology Company (Nanjing, China).
Ficoll-Hypaque solution was purchased from Haoyang Biotechnology Company (Tianjin, China). Goat serum and 4% paraformaldehyde were from Boster Biotechnology Company (Wuhan, China). VEGF-C and SDF-1 were from PeproTech. Lenvatinib (E7080) was from Selleckchem. AMD3100 was from Sigma-Aldrich. Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were from Hyclone. Complete growth medium EGM-2MV was from Lonza. 4′,6-Diamidino-2-phenylindole (DAPI) was from Beyotime Biotechnology Company (Shanghai, China). JFK (Lot number 130202) was obtained from Jilin Jinfukang Pharmaceutical Co., Ltd., (Jilin, China).